In genetically evaluating animals, researchers use theory based on nuclear genes. Higher organisms have a nucleus within their cells that contains nuclear genetic material. This has been the source of genetic material for scientists in analyzing genetic background and inheritance of animals. However, there are other organelles within the cells that have their own genetic makeup, one of which includes the mitochondria with its own DNA.
Until now, it was widely held that essentially no variation existed in mitochondrial DNA. This invention relates to the finding that there is variation, or polymorphism, within mitochondrial DNA, and these polymorphisms may be used to evaluate inheritable traits of animals.
The use of mitochondrial DNA is a significant advancement in the science of animal breeding. Current genetic improvement programs are based solely on use of nuclear genes. Classical animal breeding methods have been developed first by using mass selection and then by progressing to evaluation of lineages, pedigrees, selection index, and best linear unbiased prediction methods. Progeny testing is the breeding method used most often in cattle. All methods are based on use of nuclear genetic material. Mitochondrial genes are inherited maternally. That is, they are transmitted from a female to both sexes of progeny; males do not transmit mitochondrial genes to their progeny. Thus, in a progeny test, bull mothers pass their maternal genetic material to their sons, but these sons do not transmit this genetic material to their daughters. Both nuclear and mitochondrial genetic material contributes to the production records of bull mothers, but, because only nuclear material is transmitted through their sons to his daughters, the bull mothers may be over- or under-evaluated, depending upon the bull mother's mitochondrial contribution. If the magnitude of the cytoplasmic or mitochondrial effect is known, the pedigree estimate of the bull mother's contribution can be adjusted appropriately.
Milk volume and percentage of milk fat and milk protein (two constituents of milk, along with lactose and minerals) are economically the most important traits of dairy cattle. Reproduction and health of cattle, however, merit attention as well, because the interval between successive calvings and health costs also determine profitability of dairy cows. Effects of cytoplasmic inheritance on reproductive measures have been shown for number of days open (days from calving to next conception), days from calving to first detected estrus, first service conception rate, and number of services.
It has now been found that there are polymorphisms in mitochondrial DNA and that a method of isolating and preparing specific fragments of the mitochondrial DNA has been developed so that fragments in the mitochondrial DNA may be associated with phenotypic expression as a particular trait in the animal. This discovery has been applied specifically to a herd of dairy cattle by associating the presence of particular nucleotide substitutions in the D-loop region of mitochondrial DNA (mtDNA) with increased volume productions of milk and with greater milkfat content. Other associations are also made for important economic traits such as days open and certain health traits such as mastitis and reproductive problems.
Mitochondrial lineage influences on health differences of cattle have not been examined. Much work associating human diseases to mitochondrial DNA sequence differences has been reported. Kearns-Sayres Syndrome (KSS) and Leber's hereditary optic neuropathy (LHON) are examples of such diseases. In fact, LHON has been shown to be correlated with a single guanine-adenine transition that converts an arginine to a histidine in NADH dehydrogenase subunit 4 gene of mtDNA. Such mtDNA nucleotide sequence substitutions may affect health and other production traits in cattle.
Molecular variation in bovine mtDNA has been demonstrated through RFLP analysis and comparison of nucleotide sequences. Displacement loop (D-loop) sequences of mtDNA from 36 distinct registered maternal lineages available for this study were previously compared Lindberg, G. L. 1989. Sequence heterogenity of bovine mitochondrial DNA. Ph.D Dissertation, Iowa State University, Lib. Ames. University Microfilm order No. DA-9014925. Ann Arbor, Mich. Fifty-one sequence differences were located, including 48 single base pair (bp) substitutions, one 9-bp deletion, and two variable length poly G-C runs. Where possible, D-loops from two or more animals of the same maternal lineage were sequenced to verify accuracy of mtDNA isolation and nucleotide sequencing and to confirm constancy of mtDNA within maternal lineages, supra.
The D-loop region of mtDNA does not code for any known gene products; hence, sequence polymorphisms there would not alter specific protein chain subunits. Promoters for transcription of both heavy and light strands of mtDNA as well as the origin of heavy strand replication, however, lie within the D-loop. Thus, sequence differences in the mtDNA D-loop may alter transcription or replication rates. Moreover, such D-loop polymorphisms may serve as markers of differences elsewhere on the mtDNA genome in coding regions of genes that are associated with phenotypic expression of traits.
The primary objects of the invention are, therefore, to evaluate animals genetically by using their mitochondrial DNA by phenotypic analyses of differences between maternal lineages.
Another object of the invention is to use polymorphism in mitochondrial DNA of animals to associate the absence or presence of single or several markers with traits of economic importance including health trait importance in animals.
A further object of the invention is to determine the sequence of nucleotides in mitochondrial DNA fragments and associate presence or absence of specific nucleotide sequences of fragments with traits of economic and health importance.
A still further object is to associate polymorphism in mitochondrial DNA with its expression in the maternal lineage of an animal.
A further object is to provide for improved genetic evaluation of animals by phenotypically determining the relative worth of maternal lineages for economic traits.
Another object of the invention is to predictably isolate efficient milk producers and lineages of milk producers from dairy herds by evaluation of polymorphism in mitochondrial DNA.
Other objects of the invention will become apparent in the following disclosure.